The objective of this five year renewal program is to develop enhanced serological and virological test systems for B virus (Herpesvirus simiae) as well as other simian herpesviruses, to unambiguously identify B virus infection in humans, macaques, or other exposed nonhuman primates. We will identify structural and functional properties of B virus, and closely related herpesviruses to develop a multifaceted diagnostic system. We propose development of the following assay systems: (1) a rapid enzyme-immunoassay for serological identification of B virus specific antibodies using recombinant B virus components to replace currently required B virus propagation, (2) a rapid antigen detection system to identify the presence of replication competent B virus present in human and nonhuman primate tissues (lesional or nonlesional sites), and (3) molecular analysis using in situ hybridization techniques to identify primates with potential for reactivation of latent B virus. To accomplish the development of these systems we propose the following specific aims; (1) identify the polypeptides/glycoproteins of B virus to which human (n=6) and macaque (n=50) virus hosts direct a specific humoral response (igM and igG), (2) identify these B virus polypeptides(s)/glycoprotein(s) which contain the greatest abundance of B virus specific sequences or epitopes, (3) purify these B virus polypeptides(s)/glycoproteins(s) and subsequently, use the selected epitopes to enhance monoclonal antibody production, (4) identify the polypeptide/glycoprotein specificity of produced polyclonal monospecific antibodies and monoclonal antibodies and use these antibody reagents for purification of selected, specific, immunoreactive epitopes, (5) identify B virus specific loned DNA fragments from our B virus genomic library, (6) express these fragments in a high efficiency Autographa californica vector system, (7) develop rapid assay(s) systems using these recombinant expression products, (8) use the specific DNA fragments to identify optimum in situ probes for localization of B virus in both infected cells in culture as well as from directly sampled tissues, and (9) develop serological and virological assay systems utilizing the selected molecularly characterized B virus components from aims 1-8 for development of rapid and analytical B virus diagnostics in humans and macaques (or other exposed nonhuman primate species), including trial of these reagents to: a) identify risk factors for B virus infection and detectable shedding events within a defined breeding group of rhesus monkeys located at the California Primate Research Center and b) correlate the presence of B virus antibody, detectable B virus shedding, and latent B virus infections within trigeminal and lumbosacral dorsal root ganglia of rhesus monkeys from this population. The results of the research proposed in this renewal application will provide the basis for implementing the safest possible operating standards for NIH and other federally supported animal resource colonies and their personnel. Adoption of these standards will enhance management, prevention and control of B virus infections in humans and other primate species. Development of these diagnostic systems is especially important in preventing further zoonotic disease in humans now that macaque usage is increasing in response to the applicability of this species in AIDS research.